Whooping cough is caused by an infection of the respiratory tract with Bordetella pertussis bacteria. This disease is effectively controlled by the current vaccine which consists of killed whole B. pertussis cells. Though efficacious, the present vaccine produces unacceptable side effects. The major protective antigen in whooping cough vaccines is pertussis toxin. Chemically "inactivated" pertussis toxin vaccines have been produced with reduced side effects and reasonable efficacy, however, residual activity may exist. Using site-specific DNA mutagenesis, we modified E. coli subclones of the pertussis toxin SI subunit and then used these constructs to replace the chromosomal copy of the toxin gene in B. pertussis strain 3779. The resulting new strain produces a fully genetically detoxified form of pertussis toxin which is strongly immunoprotective and can be used as a vaccine antigen without chemical inactivation. In a recently completed NIAID-supported clinical trial in Sweden and Italy, pertussis toxin emerged as an essential component of any new whooping cough vaccine. One of the major acellular pertussis products used in this trial was a mutant version of pertussis toxin that was developed from basic research generated through this intramural research project. Molecular studies are currently underway in our laboratory to develop high yield B. pertussis strains to enhance expression of pertussis toxin for use in acellular and conjugate vaccine manufacture. In addition, we are developing genetic strategies designed to produce a new strain of B. pertussis with significantly reduced virulence factors. Further work is underway to apply a similar approach to cholera and expression of vaccine antigens in plant systems.